Aureolic acid-type anticancer agents, such as mithramycin (MTM) or chromomycin (CMM), are potent anticancer drugs with a unique mode-of-action. They inhibit the growth of cancer cells by cross-linking GC-rich DNA thereby shutting down specificity-protein (Sp)-dependent pathways towards various proto-oncogenes including c-myc and c-src, the latter being associated with the unique hypocalcemic activity found for these drugs. Particularly, MTM is important, and has become a popular biochemical tool to study Sp-dependent signal transduction pathways, but due to its toxic side effects is rarely used as anticancer agent, except for the treatment of tumor hypercalcemia refractory to other chemotherapy. However, MTM was recently identified as a potential lead drug against neurological diseases, arthritis, and for the treatment of hematologic disorders. All these new applications require only very small, less toxic concentrations of the drug, although the mode-of- action in these contexts remains obscure. MTM's biosynthesis has been studied intensely during the previous funding period of this research project, and consequently pursued combinatorial biosynthetic efforts revealed various biosynthetic intermediates and new MTM-analogues. Two of these analogues, MTM SK and MTM SDK, showed a much better anticancer activity profile with a greatly improved therapeutic index than MTM itself. These new drugs deserve further investigations. During the previous biosynthetic studies biosynthetic intriguing and interesting key enzymes were discovered, which need to be further investigated, particularly oxygenase MtmOIV, Ketoreductase MtmW, glycosyltransferases MtmGIV, MtmGIII, MtmGII, MtmGI, and other, early acting post-polyketide synthase tailoring oxygenases and reductases. The goal is to understand the role and mechanisms of these enzymes in the MTM biosynthesis, and to optimize them for the engineering of novel MTM derivatives. It is planned to (a) further investigate unclear biosynthetic steps and mechanisms of the MTM and CMM pathways and to generate new MTM analogues applying combinatorial biosynthesis, (b) to analyze intriguing oxygenases and reductases, (c) to investigate and improve the substrate specificity of glycosyltransferases, (d) to study in vitro and in vivo MTM SK, MTM SDK and other promising MTM analogues developed during the project. PUBLIC HEALTH RELEVANCE: The proposed work aims to develop and refine a new generation of aureolic acid type natural product analogues with significantly diminished toxicity that will be useful mainly as anticancer drugs, but also as drugs to treat neurological diseases, arthritis and hematologic disorders. To enable the production of these fine-tuned drugs through combinatorial biosynthesis, in-depth research of the biosynthetic machinery including key enzymes will be explored.